CN116377096A - Helicobacter pylori clarithromycin drug resistance analysis kit and detection method - Google Patents
Helicobacter pylori clarithromycin drug resistance analysis kit and detection method Download PDFInfo
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Abstract
The invention discloses an allele-specific primer of an analysis kit for helicobacter pylori clarithromycin resistance, which comprises a primer designed for a helicobacter pylori clarithromycin resistance-related gene, wherein the helicobacter pylori clarithromycin resistance-related gene is selected from helicobacter pylori 23S, the detection sites are A2143G, A2142C, A2142G and non-genetic mutation sites, the A2143G, A2142C and A2142G sites are clarithromycin resistance types, and the non-genetic mutation is clarithromycin sensitivity type; also disclosed are an assay kit for resistance diagnosis of helicobacter pylori clarithromycin and a diagnostic kit for resistance diagnosis, a fluorescent quantitative PCR reaction system matched with the assay kit, a detection method of resistance of helicobacter pylori clarithromycin, a method for detecting the resistance of helicobacter pylori in non-disease diagnosis and treatment and application of the detection method in research and development and/or screening of medicines, and the kit can inform patients whether the patients have double infection of two strains of clarithromycin sensitive type helicobacter pylori and drug resistant type helicobacter pylori at the same time and provide accurate clinical medication information.
Description
Technical Field
The invention relates to the field of medicine and biotechnology, in particular to an analysis kit for detecting drug resistance of helicobacter pylori clarithromycin by using allele specific primers and a drug resistance detection method.
Background
Helicobacter pylori (Helicobacter Pylori, h.pyrri) survives the human stomach and is one of the most common bacterial pathogens, and most of the world's population is infected with helicobacter pylori. Helicobacter pylori infection may cause chronic gastritis and peptic ulcer, and severe cases develop gastric cancer and gastric mucosa-associated lymphoid tissue lymphoma. With the widespread use of antibiotics, helicobacter pylori resistant strains are increasing, and the resistance of helicobacter pylori to antibiotics has become the most leading cause of the reduction of eradication rate, which not only brings difficulty to clinical treatment, but also increases the economic burden of patients, resulting in great waste of medical resources. Drug resistance detection is a precondition for implementing personalized treatment, can avoid repeated use of antibiotics which have developed drug resistance, and selects effective antibiotics according to drug susceptibility experiments. At present, a plurality of methods for detecting the drug resistance of helicobacter pylori antibiotics exist, the traditional method for detecting the drug resistance of helicobacter pylori antibiotics is mainly limited to drug sensitive tests, but E-test consumables are expensive, a paper sheet diffusion method lacks effective judgment standards, and an agar dilution method has higher requirements on operation technology; moreover, the drug susceptibility test does not identify the site of the mutation in the gene of the H.pyri strain. In addition, helicobacter pylori has strict requirements on culture, and grows slowly under microaerophilic conditions, so that the helicobacter pylori is not suitable for routine clinical development. Such as: (1) and (3) separating, culturing and identifying: after helicobacter pylori is cultured into bacterial colonies, the bacterial colonies are identified through biochemical reaction. As the helicobacter pylori culture needs microaerophilic conditions and has strict requirements on nutritional conditions, false negative is extremely easy to cause, the sensitivity is only 40-70%, and the detection rate is low. And the helicobacter pylori culture requires a certain time, which is unfavorable for rapid diagnosis. (2) UBT: according to different markers, the kit can be divided into 13C-UBL and 14C-UBL, and has wide clinical application and low technical requirements. The defect is high cost, easy influence by acid inhibitor and antibacterial drug and low sensitivity. (3) Immunological examination: detecting helicobacter pylori antigen in feces or helicobacter pylori antibody in serum. Has the advantages of high speed and no damage, and has the defect of being incapable of judging the current infection or the past infection. (4) Histopathological section staining method: the method is to take a biopsy of a patient from a gastroscope, and observe helicobacter pylori in the tissue after staining. The method has the advantages of capability of simultaneously carrying out pathological diagnosis of gastric mucosa and higher specificity and sensitivity. However, the method is obviously affected by helicobacter pylori load, is complex in operation and time-consuming, and is not suitable for detecting a large-flux sample. With the development of molecular biology techniques, techniques such as PCR, fluorescence in situ hybridization, gene chip, DNA sequencing, etc. have been used for the detection of helicobacter pylori antibiotic resistance, including: common PCR, oligonucleotide probe hybridization, gene chip, sequencing, etc. All of the above methods of examination are characterized, but do not allow simultaneous detection of double infections of both drug resistant and sensitive strains in one sample. We have found that the simultaneous infection of patients with H.pylori resistant and susceptible strains is common using this allele-specific fluorescent PCR technique designed by the company (see samples 22 and 26 in FIG. 2).
In helicobacter pylori infection, especially in the treatment of pediatric helicobacter pylori infection, clarithromycin is one of the important first-choice drugs. However, current infections with clarithromycin resistant strains account for almost one third of all helicobacter pylori patients, and present significant difficulties in clinical treatment, resulting in prolonged illness. It was found by study that the pharmaceutically acceptable target site of clarithromycin was H.pylori ribosomal 23S, and that drug resistance occurred by A to G or A to C mutation at nucleotide 2142 or A to G mutation at nucleotide 2143 of the H.pylori 23S gene. 2143 site A to G mutations are most common and 2142 site A to C mutations cause the strongest drug resistance.
Currently, some companies and research and development institutions mainly apply gene sequencing and TaqMan probe method technology to detect the gene mutation related to clarithromycin resistance. The mutation gene locus can be accurately known by gene sequencing, but the steps are relatively more and the time is long; the TaqMan probe method can detect whether a gene mutation exists in a probe coverage area (about 10 nucleotides), but specific mutation points and mutation nucleotides cannot be known. In case the patient is infected with both clarithromycin-sensitive and drug-resistant helicobacter pylori, neither gene sequencing nor TaqMan probe technology is clearly informed.
In PCR, the amplification direction is from the 5' end to the 3' end of the primer, and whether the first nucleotide at the 3' end of the primer completely matches with the amplified template is one of the keys to whether the PCR can start to run smoothly. When the template binding site of the first nucleotide at the 3' end of the primer is located at the nucleotide mutation point (allele) on the gene, if perfect combination (t=a, c=g) is performed, PCR can be smoothly performed under a proper temperature condition; if there is imperfect bonding (t=c, t=g, a=c, a=g), PCR operation can be greatly affected. PCR detection designed by using the perfect or imperfect combination of the 3' end of the primer and the gene detection site to influence the PCR operation is called gene allele specific PCR.
The existing molecular biology technology for detecting helicobacter pylori antibiotic drug resistance also has the defects of high cost, difficult clinical popularization and the like, so that development of a rapid, accurate, convenient and economic multi-gene detection method is needed to synchronously complete helicobacter pylori identification and multi-site drug resistance analysis, so that a novel helicobacter pylori detection and individuation helicobacter pylori eradication mode based on drug resistance analysis is explored, the clinical reasonable application of antibiotics is better guided, the appearance of drug-resistant strains is reduced, and the economic burden of patients is reduced. There is no method for simultaneously detecting clarithromycin resistance and helicobacter pylori infection based on gene allele specific PCR technology.
Disclosure of Invention
In order to solve the problems in the prior art, the invention provides a technical scheme, namely a helicobacter pylori clarithromycin drug resistance analysis kit and a drug resistance detection method, which are characterized by applying the characteristic of allele-specific PCR, designing primers respectively aiming at clarithromycin drug resistant strains A2143G, A2142C, A2142G and 4 groups without gene mutation (clarithromycin sensitivity), accurately detecting whether helicobacter pylori in a patient sample is the clarithromycin drug resistant strain, exact gene mutation sites and mutation nucleotides through simple fluorescent quantitative PCR under the same experimental conditions, and informing patients whether clarithromycin sensitivity and drug resistant helicobacter pylori double infection exist at the same time, and providing accurate information for clinical medication. The allele-specific primer designed by the invention has the PCR amplification and drug-resistant specific site detection effects, and no additional probe is required.
In one aspect, the invention provides an assay kit for detecting resistance to clarithromycin in helicobacter pylori using an allele-specific primer, comprising: the primer comprises a primer designed for a helicobacter pylori clarithromycin resistance-related gene, wherein the helicobacter pylori clarithromycin resistance-related gene is selected from helicobacter pylori 23S, detection sites are A2143G, A2142C, A2142G and a non-gene mutation site, the A2143G, A2142C and the A2142G site are clarithromycin resistance types, and the non-gene mutation is clarithromycin sensitivity type.
As a preferred embodiment, the nucleotide sequences of the upstream primer and the downstream primer for detecting the clarithromycin resistant strain A2143G are shown as SEQ ID NO. 1 and SEQ ID NO. 2, respectively;
the nucleotide sequences of the upstream primer and the downstream primer for detecting the clarithromycin drug-resistant strain A2142C are respectively shown as SEQ ID NO. 3 and SEQ ID NO. 4;
the nucleotide sequences of the upstream primer and the downstream primer for detecting the clarithromycin drug-resistant strain A2142G are respectively shown as SEQ ID NO. 5 and SEQ ID NO. 6;
the nucleotide sequences of the upstream primer and the downstream primer of the primer for detecting the non-genetic mutation are respectively shown as SEQ ID NO. 7 and SEQ ID NO. 8;
primer and probe sequences are shown in table 1:
as a preferred embodiment, the forward primer is a universal primer, and the base pair at the 3' -end of the reverse primer is located at a mutated base or allele point of the gene on the template; the forward universal Tag sequence is: 5'-AGGTGACACTATAGAATA-3' the primers designed for the clarithromycin related gene include 4 reverse primers; designed for 1 wild-type gene and 3 mutant genes, respectively.
In a second aspect, the present invention provides a reagent for analysis of resistance to helicobacter pylori clarithromycin or a kit for analysis of resistance to helicobacter pylori clarithromycin comprising the primer.
As a preferred embodiment, the kit may further comprise any one or several of the following reagents: buffer solution, DNA polymerase, fluorescent marker, dNTPs and DNA polymerase activator.
As a preferred embodiment, any one or more of the following agents are also included: qPCR buffer solution, UDGase, taq DNA polymerase, fluorescent dye, dNTP and primer.
In a third aspect, the present invention provides a diagnostic kit for diagnosis of resistance to helicobacter pylori clarithromycin, comprising a nucleic acid extraction kit and an assay kit as described in the second aspect.
The fourth aspect of the present invention is to provide a fluorescent quantitative PCR reaction system for use with the assay kit, the reaction system comprising:
the fluorescent quantitative PCR amplification system comprises the following components:
the reagent is from the Hieff qPCR SYBR Green Master Mix (No Rox) kit of Yeasen company. The prepared qPCR amplification system is uniformly mixed, and after short low-speed centrifugation, the mixture is placed in a Bo-Japanese fluorescent quantitative pcr instrument for amplification according to the following conditions:
in a fifth aspect, the present invention provides a method for detecting resistance to clarithromycin of helicobacter pylori, comprising the steps of:
extracting helicobacter pylori nucleic acid;
performing fluorescent quantitative PCR amplification on clarithromycin resistance related genes in the extracted nucleic acid by using the analysis kit;
wherein, in the fluorescence quantitative PCR amplification reaction process, primers designed for clarithromycin drug resistance genes in the kit are simultaneously used;
wherein, the fluorescent quantitative PCR amplification reaction process comprises the following steps:
degrading the template containing UAT at 25 ℃ for 5min;
denaturation at 95℃for 5min;
denaturation at 95℃for 10s, annealing at 56℃for 10s, and extension at 60℃for 20min; this step was performed 45 cycles;
displaying a melting curve;
the reaction was completed.
The method for detecting the resistance of helicobacter pylori antibiotics based on multiple genes is preferably a method for the purpose of non-disease diagnosis and treatment, wherein the resistance is caused by 3 point mutations in gene 23S; the 3 point mutations are A2143G point mutations, A2142C point mutations and A2142G point mutations.
A sixth aspect of the invention provides an electronic device comprising a processor and a memory, the memory storing a plurality of instructions, the processor being for reading the instructions and performing the method of the fifth aspect.
A seventh aspect of the invention provides a computer readable storage medium storing a plurality of instructions readable by a processor and for performing the method according to the fifth aspect.
The eighth aspect of the present invention provides the multi-gene-based helicobacter pylori antibiotic resistance detection method, which can be applied to drug development and/or screening, wherein the resistance is caused by 3 point mutations in gene 23S; the 3 point mutations are A2143G point mutations, A2142C point mutations and A2142G point mutations.
The invention has the beneficial effects that:
the detection and analysis of 3 gene mutation points related to the drug resistance of helicobacter pylori clarithromycin on helicobacter pylori ribosome 23S comprise 4 independent allele-specific fluorescence PCR, and the detection method is aimed at A2143G, A2142C, A2142G and the non-gene mutation points, wherein the A2143G, A2142C and the A2142G points are clarithromycin drug resistant types, the non-gene mutation is clarithromycin sensitive type, and can simultaneously realize rapid identification and analysis of the infection of the clarithromycin drug resistant strain and the clarithromycin sensitive strain of helicobacter pylori, the sensitivity can reach more than 90%, the accuracy can also reach 90%, and the defects of long time consumption, low positive rate, high cost and the like of the traditional culture method and drug-sensitive detection are avoided.
Drawings
FIG. 1 is a schematic diagram showing the detection results of different samples of clarithromycin resistant strains A2143G, A2142C, A2142G and 4 groups of primers without gene mutation (clarithromycin sensitivity);
FIG. 2 shows the detection of helicobacter pylori infection, clarithromycin resistant strain infection and clarithromycin sensitive strain double strain infection in a patient by analyzing the difference between melting curve and amplification CT value of 4 groups of fluorescent PCR amplification; and detecting the gene-detection result comparison table of the infection of different clarithromycin drug-resistant strains;
FIG. 3 is a graph showing the relationship between the cycle number and fluorescence intensity of 4 reverse primers (respectively for 1 Wild-type gene Wild and 3 mutant genes Alle2142C resistance, alle2142G resistance and Alle2143G resistance) included in primers designed for clarithromycin related genes under the condition of HP sensitive strain infection provided by the invention;
FIG. 4 is a graph showing the relationship between the cycle number and fluorescence intensity of 4 reverse primers (respectively for 1 Wild-type gene Wild and 3 mutant genes Alle2142C resistance, alle2142G resistance and Alle2143G resistance) included in primers designed for clarithromycin related genes under the condition of HP drug-resistant strain infection provided by the invention;
FIG. 5 shows the relationship between the cycle number and fluorescence intensity of 4 reverse primers (respectively for 1 Wild-type gene Wild and 3 mutant genes Alle2142C resistance, alle2142G resistance and Alle2143G resistance) included in the primers designed for the clarithromycin related gene under the condition of mixed infection of HP drug-resistant sensitive strains;
FIG. 6 is a graph showing the relationship between the cycle number and fluorescence intensity of 4 reverse primers (respectively for 1 Wild-type gene Wild and 3 mutant genes Alle2142C resistance, alle2142G resistance and Alle2143G resistance) included in the primers designed for clarithromycin related gene under the non-HP infection condition provided by the present invention;
fig. 7 is a schematic structural diagram of an embodiment of an electronic device according to the present invention.
Detailed Description
In order to better understand the above technical solutions, the following detailed description will be given with reference to the accompanying drawings and specific embodiments.
The method provided by the invention can be implemented in a terminal environment, and the terminal can comprise one or more of the following components: processor, memory and display screen. Wherein the memory stores at least one instruction that is loaded and executed by the processor to implement the method described in the embodiments below.
The processor may include one or more processing cores. The processor connects various parts within the overall terminal using various interfaces and lines, performs various functions of the terminal and processes data by executing or executing instructions, programs, code sets, or instruction sets stored in the memory, and invoking data stored in the memory.
The Memory may include random access Memory (Random Access Memory, RAM) or Read-Only Memory (ROM). The memory may be used to store instructions, programs, code, sets of codes, or instructions.
The display screen is used for displaying a user interface of each application program.
In addition, it will be appreciated by those skilled in the art that the structure of the terminal described above is not limiting and that the terminal may include more or fewer components, or may combine certain components, or a different arrangement of components. For example, the terminal further includes components such as a radio frequency circuit, an input unit, a sensor, an audio circuit, a power supply, and the like, which are not described herein.
1. Material
1.1. Study object
Clarithromycin resistant strain A2143G, two isolated Clarithromycin resistant strain A2143G clinical strains.
1.2. Materials and reagents
1.3. Apparatus and device
2. Experimental method
2.1. Nucleic acid extraction
The helicobacter pylori nucleic acid extraction is carried out by adopting a method for extracting the biological ExoNA nucleic acid by using a Rakah biological ExoNA nucleic acid extraction kit according to the specification, and the specific steps are as follows:
1. the tissue homogenate was transferred to a 1.5 ml centrifuge tube (kit provided) cleaned of ribonuclease free (DNase/RNase free), centrifuged at 5500 rpm at room temperature for 10-15 minutes;
2. the tissue homogenate was drawn 300. Mu.l and transferred to another (DNase/RNase free) 1.5 ml centrifuge tube (provided by kit). The tissue homogenate is drawn without disturbing the centrifugal pellet;
3. adding 300 microliter of nucleic acid lysate and 10 microliter of magnetic beads, and fully mixing for 10 minutes in an upside-down manner;
4. centrifuging at 3000 rpm for 5min, and removing all residual liquid in the tube by using a pipette;
5. 500. Mu.l of a washing solution (ethanol was added before use) was added, and the precipitated beads were sufficiently suspended (several times with short shaking) and turned upside down for 3 minutes, and the nucleic acid precipitate was sufficiently washed.
6. Centrifuging at 1500 rpm for 5 minutes, carefully decanting the supernatant;
7. repeating the step 6 and the step 7, and removing all residual liquid in the tube by using a liquid-transferring gun;
8. opening a tube cover, covering with clean filter paper, and air-drying the precipitated magnetic beads at room temperature for 5 minutes;
9. 50. Mu.l of nucleic acid eluate was added, and { short duration shaking several times } beads were sufficiently suspended, after 5 minutes of suspension, centrifuged at 5000 rpm for 5 minutes, and the centrifuged nucleic acid-containing supernatant was transferred to another (DNase/RNase free) centrifuge tube for 5 minutes.
PCR reaction
2.2.1. Primer design
4 sets of primers were designed for clarithromycin resistant strains 2143G,2142C,2142G and no gene mutation (clarithromycin sensitive). Respectively adding a forward universal Tag sequence at the 5' end of the forward primer:
5'-AGGTGACACTATAGAATA-3', the reverse universal Tag sequence 5'-GTACGACTCACTATAGGGA-3' is added to the 5' end of the reverse primer.
After optimization, the sequences of the multiplex PCR primers obtained for each gene are shown in Table 1.
TABLE 1 multiplex PCR primer sequences
The primers were synthesized to give allele-specific primers, as detailed in Table 2.
TABLE 2 allele-specific primers
2.2.2.PCR reactions
The allele-specific PCR reaction system was 20. Mu.l, and was prepared according to the following ratio:
the multiplex PCR reaction conditions were: pretreatment at 25℃for 5 min-pre-denaturation at 95℃for 5 min- (denaturation at 95℃for 10s, annealing at 56℃for 10s, extension at 60℃for 20 s) was repeated for 45 cycles, and then a melting curve was displayed.
After the reaction was completed, the mixture was stored at 4 ℃.
2.2. Agarose electrophoresis verification
Instrument and reagents: the same as 1.3.
Agarose electrophoresis-verified electrophoresis patterns of clinical clarithromycin drug-resistant strains A2143G, A2142C, A2142G and no gene mutation (clarithromycin sensitivity) were obtained.
3. Results
FIG. 1 shows the results of detection of different samples for clarithromycin resistant strains A2143G, A2142C, A2142G and 4 sets of allele-specific primers without gene mutation (clarithromycin sensitive type).
Referring to FIG. 1, the results of detecting Clarithromycin resistant strains by the gene allele-specific PCR technique, in which 6 PCR amplified fragments were all the same in size, identical in design, and free of non-specific bands, revealed that the efficiency and specificity of the primers were excellent. The difference of the brightness of the bands shows that the efficiency of the allele primers is different when the PCR amplification is performed on the gene point mutation related to the drug resistance, namely the difference of fluorescent quantitative PCR CT values, and can be used as a theoretical basis for distinguishing the HP clarithromycin drug resistant strain.
As can be seen from the above examples and FIG. 1, 4 independent sets of allele-specific fluorescent PCR results showed corresponding target bands in the target fragment region successively, indicating good primer design, high amplification efficiency and good specificity. The sensitivity of the kit for detecting the clarithromycin drug resistance is 94.4%, the specificity is 92.6%, and the accuracy is 93.3%.
Referring to FIG. 2, by analyzing the differences between the melting curves and the amplification CT values of the 4-group fluorescent PCR amplifications, it was possible to detect whether a patient had H.pylori infection, whether a clarithromycin resistant strain infection, and whether a clarithromycin resistant strain and a clarithromycin sensitive strain infection were present in vivo. The detection of the infection of different clarithromycin resistant strains shows good application prospect.
FIGS. 3-6 illustrate differences in the efficiency of PCR amplification directed by allele-specific primers (including 4 reverse primers for 1 Wild-type gene Wild-type and 3 mutant genes, ale 2142C resistance, ale 2142G resistance, and ale 2143G resistance, respectively) designed based on the mutation points of the clarithromycin resistance-related genes, respectively, for samples of HP-sensitive strain infection, HP-resistant strain cocktail infection, and non-HP infection, respectively, wherein:
(1) As shown in FIG. 3, the amplification efficiency of the primer of the Wild type gene Wild is highest for HP sensitive strain infection, and completely accords with the characteristic that the gene belongs to sensitive strain infection;
(2) As shown in FIG. 4, the amplification efficiency of the Alle2143G drug-resistant gene primer is highest for HP drug-resistant strain infection, and completely accords with the characteristic of the gene belonging to the drug-resistant strain infection;
(3) As shown in FIG. 5, for mixed infection of HP drug-resistant sensitive strains, the amplification efficiency of the wild-type gene primer and the Alle2143G drug-resistant gene primer is similar and far higher than that of Alle2142G and Alle2142C, and is completely consistent with the infection characterization of the genes belonging to sensitive and drug-resistant strains.
(4) As shown in FIG. 6, the amplification efficiency of the 4 sets of allele-specific primers was also low for non-HP infection, which was fully satisfactory for practical use.
It can be proved that the invention comprises an allele-specific primer designed for the gene mutation related to the resistance of the helicobacter pylori clarithromycin, wherein the gene related to the resistance of the helicobacter pylori clarithromycin is selected from helicobacter pylori 23S, the detection sites are A2143G, A2142C, A2142G and no gene mutation sites, the A2143G, A2142C and the A2142G sites are clarithromycin resistant types, and the theory that no gene mutation is clarithromycin sensitive (namely Wild type gene Wild) completely accords with the test result.
The invention also provides a memory storing a plurality of instructions for implementing the method according to the first embodiment.
As shown in fig. 7, the present invention further provides an electronic device, including a processor 301 and a memory 302 connected to the processor 301, where the memory 302 stores a plurality of instructions, and the instructions may be loaded and executed by the processor, so that the processor can execute the method according to the first embodiment.
While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. It is therefore intended that the following claims be interpreted as including the preferred embodiments and all such alterations and modifications as fall within the scope of the invention. It will be apparent to those skilled in the art that various modifications and variations can be made to the present invention without departing from the spirit or scope of the invention. Thus, it is intended that the present invention also include such modifications and alterations insofar as they come within the scope of the appended claims or the equivalents thereof.
Claims (10)
1. An assay kit for resistance to helicobacter pylori clarithromycin, comprising: the allele-specific primer comprises a primer designed for a helicobacter pylori clarithromycin resistance-related gene, wherein the helicobacter pylori clarithromycin resistance-related gene is selected from helicobacter pylori 23S, the detection sites are A2143G, A2142C, A2142G and no-gene mutation sites, the A2143G, A2142C and the A2142G sites are clarithromycin resistance types, and no-gene mutation is clarithromycin sensitivity type.
2. An assay kit for clarithromycin resistance to helicobacter pylori as claimed in claim 1, which comprises: the nucleotide sequences of the upstream primer and the downstream primer for detecting the clarithromycin drug-resistant strain A2143G are respectively shown as SEQ ID NO. 1 and SEQ ID NO. 2;
the nucleotide sequences of the upstream primer and the downstream primer for detecting the clarithromycin drug-resistant strain A2142C are respectively shown as SEQ ID NO. 3 and SEQ ID NO. 4;
the nucleotide sequences of the upstream primer and the downstream primer for detecting the clarithromycin drug-resistant strain A2142G are respectively shown as SEQ ID NO. 5 and SEQ ID NO. 6;
the nucleotide sequences of the upstream primer and the downstream primer of the primer for detecting the non-genetic mutation are respectively shown as SEQ ID NO. 7 and SEQ ID NO. 8;
primer and probe sequences are shown in table 1:
3. the kit for analysis of resistance to clarithromycin of helicobacter pylori according to claim 2, wherein the forward primer is a universal primer and the base pair at the 3' -end of the reverse primer is located at a mutated base or an allelic point of the gene on the template; the forward universal Tag sequence is: 5'-AGGTGACACTATAGAATA-3' the primers designed for the clarithromycin related gene include 4 reverse primers; designed for 1 wild-type gene and 3 mutant genes, respectively.
4. A clarithromycin resistance assay kit for helicobacter pylori or a clarithromycin resistance assay kit for helicobacter pylori comprising the primer of any one of claims 1 to 3.
5. The assay kit of claim 4, further comprising any one or more of the following reagents: qPCR buffer solution, UDGase, taq DNA polymerase, fluorescent dye, dNTP and primer.
6. A kit for diagnosis of resistance to helicobacter pylori clarithromycin comprising a nucleic acid extraction kit and an assay kit as defined in any one of claims 4 to 5.
7. A fluorescent quantitative PCR reaction system for use with an assay kit according to any one of claims 4 to 5, said reaction system comprising:
the fluorescent quantitative PCR amplification system comprises the following components:
The above reagent is from Yeasen company
qPCR SYBR Green MasterMix (No Rox) kit; the prepared qPCR amplification system is uniformly mixed, and after short low-speed centrifugation, the mixture is placed in a Bo-Japanese fluorescent quantitative pcr instrument for amplification according to the following conditions:
8. a method for detecting drug resistance of helicobacter pylori clarithromycin comprises the following steps:
extracting helicobacter pylori nucleic acid;
performing fluorescent quantitative PCR amplification of clarithromycin resistance related gene in the extracted nucleic acid using the assay kit of any one of claims 1-3;
wherein, in the fluorescence quantitative PCR amplification reaction process, primers designed for clarithromycin drug resistance genes in the kit are simultaneously used;
wherein, the fluorescent quantitative PCR amplification reaction process comprises the following steps:
degrading the template containing UAT at 25 ℃ for 5min;
denaturation at 95℃for 5min;
denaturation at 95℃for 10s, annealing at 56℃for 10s, extension at 60℃for 20s; this step was performed 45 cycles;
displaying a melting curve;
the reaction was completed.
9. Use of the method for detecting resistance to a multigene-based helicobacter pylori antibiotic according to claim 8 in a method for the purpose of non-disease diagnosis and treatment, wherein the resistance is caused by 3 point mutations in gene 23S; the 3 point mutations are A2143G point mutations, A2142C point mutations and A2142G point mutations.
10. Use of the multi-gene based helicobacter pylori antibiotic resistance detection method as defined in claim 8 in the development and/or screening of a drug, wherein the resistance is caused by 3 point mutations in gene 23S; the 3 point mutations are A2143G point mutations, A2142C point mutations and A2142G point mutations.
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